Modulation of MMP-2 and -9 secretion by cytokines, inducers and inhibitors in human melanoma A-2058 cells
- Authors:
- Published online on: April 24, 2017 https://doi.org/10.3892/or.2017.5597
- Pages: 3681-3687
Abstract
Introduction
Cancer of the skin is by far the most common of all cancers. Melanoma accounts for only ~1% of skin cancers but causes a large majority of skin cancer deaths, due to metastasis to other areas of the body, such as lymph nodes, lungs, liver, brain or bone. Though often curable in its early stages, metastatic malignant melanoma is an extremely aggressive cancer with no current viable treatment. The American Cancer Society estimates that 76,380 new melanomas will be diagnosed (~46,870 in men and 29,510 in women) in the United States for 2016. Approximately 10,130 people are expected to die of melanoma (~6,750 men and 3,380 women). The rates of melanoma have been rising for the last 30 years (1).
Thus, any successful treatment for melanoma has to target metastasis, which is dependent upon degradation of the extracellular matrix (ECM), which, when intact, acts as a barrier to block cancer cell invasion (2–4). Clinical and experimental studies have demonstrated that elevated levels of matrix metalloproteinases are associated with rapid progression of metastatic melanoma (5,6). MMP activity is regulated by and dependent upon environmental influences from surrounding stroma cells, ECM proteins, systemic hormones and other factors. Inflammation has been reported to drive cancer progression (7–9). Inflammatory cytokines such as interleukin (IL)-1β play significant roles in inflammation driven melanoma growth and progression (10). In the present study we investigated the effects of selected cytokines, inducers and inhibitors affecting cancer cell metabolism on the regulation of MMP-2 and MMP-9 activities in melanoma A-2058 cell line.
Materials and methods
Materials
Human melanoma A-2058 cells were obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA). Antibiotics, penicillin and fetal bovine serum (FBS), were obtained from Gibco-BRL (Long Island, NY, USA). Twenty-four well tissue culture plates were obtained from Corning Costar Corp. (Cambrdige, MA, USA). Gelatinase zymography was performed in 10% Novex pre-cast SDS polyacrylamide gel (Invitrogen) with 0.1% gelatin in non-reducing conditions. Interleukin 1β, tumor necrosis factor-α (TNF-α), phorbol 12-myristate 13-acetate (PMA), lipopolysaccharide (LPS), doxycycline, epigallocatechin gallate (EGCG), actinomycin-D, cyclohexamide, retinoic acid and dexamethasone, were purchased from Sigma-Aldrich (St. Louis, MO, USA). The nutrient mixture (NM), prepared by VitaTech (Hayward, CA, USA) was composed of the following ingredients in the relative amounts indicated: Vitamin C (as ascorbic acid and as Mg, Ca and palmitate ascorbate) 700 mg; L-lysine 1,000 mg; L-proline, 750 mg; L-arginine, 500 mg; N-acetyl cysteine, 200 mg; standardized green tea extract (80% polyphenol), 1,000 mg; selenium, 30 µg; copper, 2 mg; manganese, 1 mg. All other reagents used were of high quality and were obtained from Sigma-Aldrich, unless otherwise indicated.
Cell cultures
Melanoma cells were grown in Dulbeccos modified Eagles medium (DMEM), supplemented with 15% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin in 24-well tissue culture plates. The cells were plated at a density of 1×105 cells/ml and grown to confluency in a humidified atmosphere at 5% CO2 at 37°C. Serum-supplemented media were removed and the cell monolayer was washed once with phosphate-buffered saline (PBS) and with the recommended serum-free media. The cells were then incubated in 0.5 ml of serum-free medium with various cytokines, mitogens, inducers and inhibitors in triplicate, as indicated: PMA (10, 25, 50 and 100 ng/ml); TNF-α and IL-1β (0.1, 1, 10 and 25 ng/ml); LPS (10, 25, 50 and 100 µg/ml); EGCG (10, 25, 50 and 100 µM) without and with PMA 100 ng/ml; doxycycline (10, 25, 50 and 100 µM) without and with PMA 100 ng/ml; NM (10, 50, 100, 500 and 1,000 µg/ml) without and with PMA 100 ng/ml, retinoic acid (50 µM); dexamethasone (50 µM); actinomycin D and cyclohexamide (2 and 4 µg/ml). The plates were then returned to the incubator. The conditioned medium from each treatment was collected separately, pooled and centrifuged at 4°C for 10 min at 3,000 rpm to remove cells and cell debris. The clear supernatant was collected and used for gelatinase zymography, as described below.
Gelatinase zymography
Gelatinase zymography was utilized because of its high sensitivity to gelatinolytic enzymatic activity and ability to detect both pro and active forms of MMP-2 and MMP-9. Upon renaturation of the enzyme, the gelatinases digest the gelatin in the gel and reveal clear bands against an intensely stained background. Gelatinase zymography was performed in 10% Novex pre-cast SDS polyacrylamide gel in the presence of 0.1% gelatin under non-reducing conditions. Culture media (20 µl) were mixed with sample buffer and loaded for SDS-PAGE with Tris-Glycine SDS buffer, as suggested by the manufacturer (Novex). Samples were not boiled before electrophoresis. Following electrophoresis the gels were washed twice in 2.5% Triton X-100 for 30 min at room temperature to remove SDS. The gels were then incubated at 37°C overnight in substrate buffer containing 50 mM Tris-HCl and 10 mM CaCl2 at pH 8.0 and stained with 0.5% Coomassie Blue R250 in 50% methanol and 10% glacial acetic acid for 30 min and destained. Protein standards were run concurrently and approximate molecular weights were determined by plotting the relative mobilities of known proteins. Gelatinase zymograms were scanned using CanoScan 9950F Canon scanner at 300 dpi. The intensity of the bands was evaluated using the pixel-based densitometer program Un-Scan-It, version 5.1, 32-bit, by Silk Scientific, Inc., (Orem, UT, USA), at a resolution of 1 Scanner Unit (1/100 of an inch for an image that was scanned at 100 dpi).
Statistical analysis
Microsoft Excel 2010 linear trend analysis was utilized to determine the linear trend analyses of the densitometry results.
Results
Inducers, mitogens and cytokines
Melanoma A-2058 expressed bands corresponding to MMP-2 and MMP-9. Table I shows the quantitative densitometry results from the effects of PMA, TNF-α, IL-1β and LPS on MMP-2 and MMP-9 expression in A-2058 cells.
Effect of PMA on A-2058 cell secretion of MMPs
On gelatinase zymography, A-2058 demonstrated strong expression of MMP-2 and slight expression of MMP-9. PMA treatment ranging from concentrations of 10–100 ng/ml showed no significant effect on MMP-2 secretion (linear trend R2=0.009) but significant dose-dependent upregulation of MMP-9 secretion with 400% that of control at 100 ng/ml (linear trend R2=0.883) (Fig. 1).
Effect of TNF-α on A-2058 cell secretion of MMPs
On gelatinase zymography, A-2058 demonstrated strong expression of MMP-2 and slight expression of MMP-9. TNF-α, used between 0.1–25 ng/ml, showed no significant overall effect on expression of MMP-2 (linear trend R2=0.037). TNF-α showed dose-dependent increased MMP-9 secretion (linear trend R2=0.876) with 200% of control at 25 ng/ml (Fig. 2).
Effect of IL-1β on A-2058 cell secretion of MMPs
Gelatinase zymography demonstrated strong expression of MMP-2 by A-2058 cells and slight expression of MMP-9. IL-1β at a concentration range of 0.1–25 ng/ml did not have significant effect on MMP-2 secretion by A-2058 cells, except at 25 ng/ml where MMP-2 level was reduced by ~40% (linear trend R2=0.529). Also, MMP-9 secretion was not modified by IL-1β except for its ~50% reduction at 25 ng/ml (Fig. 3).
Effect of LPS on A-2058 cell secretion of MMPs
Gelatinase zymography demonstrated strong expression of MMP-2 and slight expression of MMP-9 by A-2058 melanoma cells. LPS treatment between 10–100 µg/ml showed no significant effect on MMP-2 secretion (linear trend R2=0.375). However, enhanced MMP-9 secretion was observed at LPS up to 25 µg/ml that was followed by a decreased MMP-9 level (linear trend R2=0.244) (Fig. 4).
Chemical inhibitors
Table II shows the quantitative densitometry results from the effects of select chemical inhibitors doxycycline, dexamethasone, actinomycin D and cyclohexamide on MMP-2 and MMP-9 expression in melanoma A-2058 cell line.
Effect of doxycycline on A-2058 cell secretion of MMPs
Doxycycline inhibited MMP-2 secretion by A-2058 cells in a dose-dependent manner with 72% blockage at 100 µM (linear trend R2=0.843). MMP-9 secretion was not affected by doxycycline up to 50 µM, and at 100 µM it was decreased by 30% (linear trend R2=0.480). In the presence of PMA at 100 ng/ml, doxycycline downregulated the expression of MMP-2 in a dose-dependent manner, with 67% block (R2=0.8072) at 100 µM and 70% block (linear trend R2=0.6711) of MMP-9 at 100 µM (Fig. 5).
Effect of actinomycin D on A-2058 cell secretion of MMPs
Actinomycin D had moderate inhibitory effect on A-2058 MMP-2 secretion (R2=0.797) with 26 and 27% inhibition at 2 and 4 µM, respectively, as shown in Fig. 6.
Effect of cyclohexamide on A-2058 cell secretion of MMPs
Cyclohexamide had potent dose-dependent inhibitory effect on MMP-2 secretion, resulting in its inhibition by 80% at 4 µM (linear trend R2=0.727), as shown in Fig. 6.
Effect of dexamethasone on A-2058 cell secretion of MMPs
Dexamethasone had slight stimulatory effect on MMP-2, with 107% of control at 50 µM (data not shown).
Natural inhibitors
Table II shows the quantitative densitometry results, presenting the effects of natural compounds EGCG alone, the EGCG in a complex with other natural compounds (NM) and retinoic acid on MMP-2 and MMP-9 expression in melanoma A-2058 cells.
Effect of EGCG on A-2058 cell secretion of MMPs
Fig. 7 shows the effects of EGCG at 10, 25, 50 and 100 µM concentrations on MMP-2 and MMP-9 secretions by unstimulated and PMA-stimulated A2058 melanoma cells. EGCG showed inhibitory effects on MMP-2 and −9 secretions by melanoma cells in a dose-dependent manner. In the presence of 100 µM EGCG, the secretion of MMP-2 decreased by 57.4% (linear trend R2=0.697) and MMP-9 by 73.2% (linear trend R2=0.519), as shown in Fig. 7A and C. In the presence of PMA (100 ng/ml) the secretion of these enzymes was inhibited by EGCG in a dose-dependent manner. Slight inhibitory effect of EGCG on PMA-induced MMP-9 secretion was already observed at 25 µM (30% inhibition) and its total block at 100 µM (linear trend R2=0.901), as shown in Fig. 7B and D.
Effect of NM on A-2058 cell secretion of MMPs
As shown in Fig. 8A and C, NM was effective in inhibiting secretion of MMP-2 by uninduced A-2058 cells in a dose-dependent manner when used at 10, 50, 100, 500 and 1,000 µg/ml (linear trend R2=0.868) with virtual total block at 1,000 µg/ml. NM also showed dose-dependent inhibition of MMP-2 and −9 expression in PMA-treated A-2058 cells as presented in Fig. 8B and D, which showed 96% blockage of MMP-2 secretion at 1,000 µg/ml and total block of MMP-9 at 1,000 µg/ml (linear trends R2=0.889 and 0.818, respectively).
Effect of retinoic acid on A-2058 cell secretion of MMPs
Retinoic acid inhibited A-2058 MMP-2 secretion with total block at 50 µM (data not shown).
Discussion
Elevated MMP levels correlate with melanoma tumor progression, as documented in clinical studies (5,6). Thus, knowledge of MMP regulation is of importance for developing therapeutic strategies for melanoma. Nikkola et al (5) found that melanoma patients with high-serum levels of MMP-9 had significantly poorer overall survival than patients with lower serum MMP-9 levels and that high MMP-9 levels were correlated with visceral or bone metastasis and presence of liver metastases. Malaponte et al (6) found that plasma levels of MMP-2 and TGF-β in patients with primary melanoma were significantly higher than those of healthy controls and those significantly higher levels were found in patients with metastatic melanoma. Extracellular factors, such as the inflammatory cytokine IL-1β has been implicated in facilitating inflammation and melanoma tumor growth (10).
In the present study, we compared MMP secretion patterns in the presence of cytokines, PMA and LPS in melanoma A-2058 cells. In addition, we investigated the effect of inhibitors doxycycline, EGCG, NM and other compounds, such as dexamethasone, cyclohexamide, retinoic acid and agents that affect protein transcription and translation levels, such as actinomycin D. None of the tested inducers and cytokines was found to enhance MMP-2 secretion. PMA and TNF-α treatment showed potent dose-dependent upregulation of MMP-9 secretion up to 400% that of control at 100 ng/ml PMA and 200% of control at 25 ng/ml TNF-α. Surprisingly, IL-1β demonstrated no effect on MMP-9 secretion, except its decrease at 25 ng/ml and LPS showed no consistent effect, resulting in an upregulation and downregulation of MMP-9 secretion. Among tested inhibitors, chemical inhibitor doxycycline and natural inhibitors EGCG and NM with and without PMA treatment, potently downregulated the secretion of A-2058 MMP-2 and MMP-9 in a dose-dependent manner. MMP-2 secretion was potently inhibited by cyclohexamide and retinoic acid, moderately inhibited by actinomycin D and slightly stimulated by dexamethasome.
In addition to individual compounds we tested the effects of a specific nutrient mixture (NM) which has demonstrated anticancer efficacy in various in vitro and in vivo studies by affecting various mechanisms, including MMPs secretion (11). EGCG from green tea is one of its components, with green tea extract comprising ~23% total NM weight. Another component is amino acid lysine, which is a natural inhibitor of plasmin-induced proteolysis, including MMPs (12,13). Lysine also contributes to 23% of weight in NM. These and other compounds in NM (i.e. vitamin C, lysine, proline, copper, manganese and N-acetyl cysteine) are important in supporting the integrity and stability of connective tissue through various mechanisms, thereby contributing to tumor encapsulation and curtailing cancer cells spread (14–22). In addition, NM components N-acetyl cysteine and selenium have been shown to inhibit tumor cell MMP-9 and invasive activities and migration of endothelial cells through the ECM (23–25). Ascorbic acid has been documented to modulate cancer cell and tumor growth as well as to prevent metastasis (26–31) and low levels of ascorbic acid are found in cancer patients (32–34). The inhibitory effects of NM in comparison to EGCG alone on MMP-9 and MMP-2 secretion in unstimulated and PMA-stimulated A-2058 cells suggest enhancing effectiveness of EGCG when applied in a mixture with other natural compounds of similar and complementary mechanisms. The contribution of individual NM components towards inhibition of MMPs warrants further investigation.
In conclusion, our results showed that various inducers, cytokines, mitogens and inhibitors tested in this study modulate MMP-2 and −9 secretions by melanoma A-2058 cells. They suggest the clinical use of MMP inhibitors, especially potent and non-toxic ones as the nutrient mixture (NM) and its component EGCG in management of melanomas.
Acknowledgements
The present study was funded by the Dr. Rath Health Foundation (Santa Clara, CA), a non-profit organization.
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